The sodium pump is responsible for the regulation and control of fluid and electrolyte balance in cells, organs and in the entire organism. It is an integral membrane protein that utilizes as much as 40% of the energy of cells to actively transport Na and K ions against their gradients utilizing the energy of hydrolysis of cellular ATP. The pump is composed of two main subunits, the ? subunit, the catalytic subunit of about 1000 amino- acids and a ? subunit of about 300 amino-acid residues which is heavily glycosylated. The sodium pump, or Na,K-ATPase, is essential for salt and fluid regulation and is found exclusively in the basolateral membrane of polarized epithelial cells of the kidney and intestines. The planned experiments will focus on the cell physiological roles of the sodium pump, focusing on the multiple roles of the ??subunit in mammalian cells. Our previous work has led to a hypothesis of how the ? subunit facilitates release of the Na pump ?? complex from the endoplasmic reticulum and enables its delivery to the plasma membrane. We plan to test this hypothesis in the proposed studies. We will also examine the role played by the Na pump ? subunit in establishing and maintaining cell-cell contacts in epithelial cells. We have discovered conditions where the exclusive basolateral delivery of the sodium pump can be over-ridden and it is delivered to the apical surface. We plan to utilize these protocols to understand how the polarized membrane delivery is controlled. Throughout these experiments we will also probe the physiological role of ? subunit glycosylation, which is currently unknown. We have developed a regulated expression system for increasing the levels of the ? subunit in mammalian cells and RNA-based methods for its reduction. We will combine strategies from molecular biology, cell physiology, biochemistry and confocal microscopy to pursue our scientific goals. The cellular roles of the ? subunit of the sodium pump are not well understood. The sodium pump is the primary membrane protein involved in fluid and electrolyte balance and is the major target of cardiac glycosides, such as digitalis, the most widely used therapy for cardiac insufficiency. Disregulation of the sodium pump is also associated with several disease states, including hypertension and migraines. The work proposed in the current application will provide new information about how this important protein carries out its functions in cells.